Mitochondria play a unique role in the metabolism of iron. The early and late steps of porphyrin biosynthesis occur in mitochondria and culminate in the incorporation of iron into protoporphyrin IX by the enzyme ferrochelatase to form heme. Mitochondria also participate in the biosynthesis of iron sulfur clusters. Heme biosynthesis is singularly important in erythrocytes, which contain greater than 70% of a mammal's iron in the form of hemoglobin. The enzymes required for the bioassembly of heme have been well characterized for more than 30 years. Over the past several years, many of the transporter and accessory proteins involved in mammalian intercellular iron metabolism have also been described. However, proteins involved in intracellular iron and heme transport, particularly those involved in mitochondrial iron utilization, remain elusive. We have recently found a mutation in a strain of mice, flexed-tail (locus symbol f) that has a defect in erythroid mitochondrial iron metabolism manifest as pathologic iron deposits in erythroid mitochondria. This phenotype resembles a group of human disorders known as sideroblastic anemias. The flexed-tail protein, which we have named sideroflexin 1 (gene symbol Sfxn1 ), is a mitochondrial multiple transmembrane protein that is a member of a family of previously undefined eukaryotic proteins. The work proposed in this grant involves an in depth characterization of Sfxn1, its homologues, and their function, with the goal of advancing our knowledge of mitochondrial iron metabolism. We specifically propose to characterize the pattern of expression and localization of the murine sideroflexins, investigate known metabolic pathways involved in the pathogenesis of sideroblastic aniemias to see if they are altered in flexed-tail mice, and utilize screening methods in yeast to determine sideroflexin function.